Short and long term regulation of catecholamine biosynthetic enzymes by angiotensin in cultured adrenal medullary cells: Molecular mechanisms and nature of second messenger systems

The purpose of this study was to examine the effects of angiotensin on the enzyme activities and gene expression of two catecholamine synthesizing enzymes, tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT), in bovine adrenal medullary (AM) cells. Short term (15 min) incubation of cultured AM cells with 2 nM [Sar¹]angiotensin II (s¹-AII) did not increase basal secretion of catecholamines; however, longer incubations (3, 24, or 72 h) produced 4-10-fold increases. To determine whether angiotensin affects synthesis of catecholamines, the activities of TH and PNMT were examined. Incubation with s¹-AII (15-30 min) decreased the K_m of TH for its biopterine cofactor ((6R)-5,6,7,8-tetrahydro-1-biopterin dihydrochloride (BH₄)) without affecting the V_max, suggesting activation of TH. After long term incubation (72 h) the K_m value was identical to that of control, while increases in the apparent V_max were observed. PNMT activity was unaffected during a 30-min treatment with s¹-AII; however, 2-fold increases occurred after a 48-72-h incubation. s¹-AII (24 h) increased the relative abundance of TH and PNMT mRNAs, suggesting that the long term increase in enzyme activities reflected increased expression of TH and PNMT genes. Maximal increases were observed at 2 nM s¹-AII and the changes were antagonized by saralasin. Induction of TH mRNA by s¹-AII was additive to the effects of veratridine or forskolin indicating that effects of angiotensin were not due to membrane depolarization or increased cyclic AMP levels. Incubation with Ca²⁺ ionophore A23187 increased TH and PNMT mRNA levels in AM cells raising the possibility that the increase in cellular [Ca²⁺] could mediate effects of angiotensin. Angiotensin-induced increases in TH and PNMT mRNA were inhibited by nifedipine indicating involvement of voltage-dependent Ca²⁺ channels. In addition, the increases in TH, but not PNMT mRNA, were antagonized by dantrolene, which inhibits mobilization of Ca²⁺ from intracellular stores. Calmodulin involvement was suggested by the inhibition of s¹-AII induced changes in mRNA with 1 μM calmidazolium. The role of protein kinase C (PKC) was indicated by the following observations: (a) increases in the membrane-bound activity of PKC in cells incubated with s¹-AII; (b) induction of TH and PNMT mRNA with PKC-activating phorbol esters; 12-O-tetradecanoylphorbol 13-acetate and 4β-phorbol 12,13-didecanoate; (c) inhibition of the s¹-AII effects by pretreatment of cells with 12-O-tetradecanoylphorbol 13-acetate, which reduced total cellular activity of PKC and attenuated the increase in mRNAs in response to phorbol ester; and (d) inhibition of changes in mRNA by a PKC inhibitor, sphingosine. In conclusion, angiotensin was found to control the expression of genes encoding catecholamine biosynthetic enzymes. The effects of angiotensin seem to be mediated by Ca²⁺-dependent second messenger systems. By these mechanisms angiotensin may exert a long term control of the secretory functions of AM cells.